Although HIV-1 integrase remains a priority target for development of small-molecule antagonists, a high-resolution structure of the intact molecule remains elusive. Using a combination of protein footprinting and mass spectrometry, we have been successful in defining the binding site for pyridoxal phosphate on intact HIV-1 integrase. NMR studies with the polypurine tract primer of (+) strand DNA synthesis have demonstrated a change in sugar ring conformation at the PPT-U3 junction, suggesting this may be an important determinant for its recognition by the RNase H domain of HIV-1 reverse transcriptase (RT). Finally, targeted insertion of nucleoside analogs has defined regions of the nucleic acid substrate that interact with the DNA polymerase domain of Ty3 RT. An extension of this study investigated whether Ty3 RT mutants were capable of reversing analog-induced inhibition of DNA synthesis. Such biochemical complementation highlighted residues of the Ty3 thumb subdomain that contact individual bases of the template-primer duplex, illustrating the importance of nucleoside analog interference strategies.Selective 2'hydroxyl acylation analyzed by primer extension (SHAPE) examines RNA secondary structure via sensitivity of the ribose 2'OH in an unpaired configuration to acylation. Our initial SHAPE studies focused on wild-type and mutant HIV-1 Rev response elements (RREs) and subsequently examined the minimal transport element of the murine LTR-retrotransposon MusD. In parallel, we expanded this technology by developing methods to examine the structure of short RNA/DNA hybrids by mass spectrometry (SHAMS) and defining tertiary interactions via antisense-interfered SHAPE (ai-SHAPE). While structural analysis of regulatory RNAs will continue, we recognize that SHAPE is not a stand-alone technique. Chemo-enzymatic footprinting will therefore be complemented with (a) NMR spectroscopy, where the size of the RNA permits, (b) small-angle X-ray scattering, and (c) targeted Fe-EDTA-footprinting with """"""""threading intercalators,"""""""" prepared in collaboration with the CCR Chemical Biology Laboratory. A long-term goal of our Section is to examine RNA structure in the context of the viral RNA genome, and novel approaches to increase SHAPE sensitivity for in vivo footprinting will be investigated.[Corresponds to Le Grice Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]

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Sztuba-Solinska, Joanna; Le Grice, Stuart F J (2018) The ""Topological Train Ride"" of a viral long non-coding RNA. RNA Biol 15:13-16
Rausch, Jason W; Miller, Jennifer T; Le Grice, Stuart F J (2017) Reverse Transcription in the Saccharomyces cerevisiae Long-Terminal Repeat Retrotransposon Ty3. Viruses 9:
Sztuba-Solinska, Joanna; Rausch, Jason W; Smith, Rodman et al. (2017) Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins. Nucleic Acids Res 45:6805-6821
Le Grice, Stuart F J; Sztuba-Solinska, Joanna; Maccioni, Elias et al. (2017) Meeting report: Third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS). Antiviral Res 139:13-17
Shapiro, Bruce A; Le Grice, Stuart F J (2016) Advances in RNA structure determination. Methods 103:1-3
Rausch, Jason W; Sztuba-Solinska, Joanna; Lusvarghi, Sabrina et al. (2016) Novel Biochemical Tools for Probing HIV RNA Structure. Methods Mol Biol 1354:91-117
Brinson, Robert G; Miller, Jennifer T; Kahn, Jason D et al. (2016) Applying Thymine Isostere 2,4-Difluoro-5-Methylbenzene as a NMR Assignment Tool and Probe of Homopyrimidine/Homopurine Tract Structural Dynamics. Methods Enzymol 566:89-110
Rausch, Jason W; Le Grice, Stuart F J (2015) HIV Rev Assembly on the Rev Response Element (RRE): A Structural Perspective. Viruses 7:3053-75
Sherpa, Chringma; Rausch, Jason W; Le Grice, Stuart F J et al. (2015) The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication. Nucleic Acids Res 43:4676-86
Abbondanzieri, Elio A; Le Grice, Stuart F J (2014) Unraveling the gymnastics of reverse transcription through single molecule spectroscopy. AIDS Res Hum Retroviruses 30:209-10

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